Quality Assessment of Cannabidiol Rich Cannabis Extracts Purchased on the Internet Quality Assessment of Cannabidiol Rich Cannabis Extracts

Research shows that cannabis has a beneficial impact on many different health issues and, as a consequence, concentrated cannabis derived products are becoming increasingly popular. Above all, high Cannabidiol (CBD) and low Tetrahydrocannabinol (THC) extracts are aggressively marketed by many vendors via internet-based shops. As with “classical” medication or food supplements, assessment of product safety is an important issue. Because many different non-standardized production methods are used and no international accepted technical verification procedures of the final products are yet available, the quality of CBD oils generally cannot be guaranteed. In this study, cannabinoid, terpene, pesticide, solvent and metal content of 24 CBD rich extracts purchased from several internet vendors were investigated. Furthermore, consumer information given on the internet site or as package insert is also discussed. Most of the samples had substandard consumer information and CBD concentrations below the proclaimed values. Terpene concentrations were highly variable, pesticides were detected in three samples and residual extraction solvents were detected in four samples. Metals were present at low, non-toxic levels in all samples. Considering the growing popularity of medical cannabis and of online shops consumer education is important and systematic quality testing for cannabis extracts should be mandatory.

Meanwhile, the internet is becoming a popular source of medical advice and an online pharmacy for standard and exotic pharmaceuticals or food supplements [10]. Beside the ethical, legal and regulatory aspects of this trade, major challenges are public health issues such as manufacturing and distribution practice, patient information and, above all, product quality [11,12]. However, often, easy ordering, and low prices are of higher priority than product quality for buyers. Even if regulation efforts have been made, quality control of products sold on the internet remains a major concern [13]. In fact, internet drug outlets may be illegitimate or illegal [14] making quality control efforts illusionary.

CBD oils
CBD oils were ordered from 25 online vendors. Twenty-four samples were finally bought, one vendor refused to sell products to an analytical laboratory. Samples were bought from Austria, France, Germany, Great Britain, Luxembourg, Netherlands and Spain. All samples were received in good condition in three days to two weeks after ordering. They were all stored at 5°C in the dark, even if three vendors recommended storing at room temperature.

Analytical methods
Cannabinoid and terpene content were determined using validated HPLC/UV and GC/MS methods, respectively. Validation parameters included the determination of the linearity, intraday precision and accuracy, Limit of Detection (LOD) and Lower Limit of Quantification (LLOQ). The LOD was defined as a signal to noise ratio equal of above three and the LLOQ as a signal to noise ratio equal or above 10. A six-point calibration curve was established before each series of analysis. For cannabinoid dosage, 50 to 100 mg of oil were dissolved in 20mL of ethanol, homogenized and diluted again in HPLC buffer (20mM KH 2 PO 4 pH 2.18/acetonitrile, 35/65, v/v) to obtain concentrations in the 0.1mg/L range. 10µL of this solution were injected into the HPLC/UV system. Analyses were carried out on a reverse phase high performance liquid chromatography system coupled to UV detector (Dionex Ultimate 3000 RS HPLC/ UV, Thermo Fisher, Waltham, USA). Chromatographic separation was achieved on an Acclaim™ Polar Advantage II column (100mm x 2.4mm x 2.2µm). Detection was obtained using a photodiode array detector (PDA, 200-360nm). Quantification wavelength was 209nm for decarboxylated cannabinoids and 223nm for carboxylated cannabinoids. Oven temperature was set at 50°C, chromatographic conditions were developed using HPLC quality water containing 20mM KH 2 PO 4 (eluent A) and acetonitrile (eluent B). Chromatographic conditions were 39% eluent A/61% eluent B for 4min followed by a linear gradient 61-90% B in 3min. Returning to initial conditions was achieved in 0.4min. Equilibration time was 2min and the flow rate was fixed at 0.85 mL/min over the whole run.
A 6 points linear calibration curve was established before analysis of the real case samples. Absolute amounts injected were 0 (blank), 10, 25, 50, 75 and 100ng of the cannabinoids in eluent A. The coefficient of determination (r 2 ) was >0.99 and resolution was >1.5 for all cannabinoids. Coefficient of variation and accuracy bias were <15%. In real case samples, a relative standard deviation of the relative peak purity index <5% was considered for positive detection and a S/N >10 for quantification of cannabinoids. All analyses were performed in duplicate; the average of the two results was retained.
For terpene determination, 100 to 150mg of oil were dissolved in 5 or 10mL of ethyl acetate, homogenized and diluted again from 10 to 100 times to fit into calibration ranges. Butyl paraben (at a concentration of about 10mg/L) was added as internal standard to each sample before analysis. 1µL of the solution was injected into the GC/MS system (7890A gas chromatography, equipped with an automatic injector AS 7693 and coupled to a 5975C mass spectrometer, Agilent Technologies, Waldbronn, Germany). Chromatographic separation was achieved on a HP-5 MS (5% phenyl methyl siloxane) column (30m x 0.25mm x 0.25µm) with helium as vector gas. Detection was done in scan mode (m/z 50-600). Injector temperature was 250°C. Initial chromatographic conditions were 70°C for 1min then 25°C/min to 190°C and then 10°C/min to 310°C for 25min. Flow rate was fixed at 1.2mL/min over the whole run. A 6 points linear calibration curve was established before analysis of the real case samples. 1µL of solutions of 0, 20, 100, 225, 350 and 450µL of the terpenoids content in 1mL ethyl acetate (at a concentration of about 10mg/L) with 100 µL of butyl paraben as internal standard (at a concentration of about 10mg/L) was injected. r 2 was >0.98 and resolution was >1.5 for all terpenes. Coefficient of variation and accuracy bias were <15%. In real case samples, a S/N >3 was considered for positive detection and a S/N >10 for quantification. LOD was 0.2ng/µL.
Determination of residual solvent was carried out on a Head Space GC/MS system (HS/GC/MC) consisting of a 6890N gas chromatography, equipped with a head space sampler 7697A and coupled to a 5975C mass spectrometer (Agilent Technologies, Waldbronn, Germany). Chromatographic separation was achieved on a DB-624 (bonded and cross-linked) column (30m x 0.32mm x 1.80µm) with helium as vector gas. Detection was done in scan mode scanning m/z from 25 to 300. Oven temperature was fixed at 85°C.

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GC/MS injector temperature was 250°C. Initial chromatographic conditions were 40°C for 4min then 12.5°C/min to 160°C and then 100°C/min to 220°C for 0.8 min. Flow rate was fixed at 2.48ml/min over the whole run. A mass of 100 to 150mg of oil was introduced in 10mL head space vial with 100µL of a 1-butanol solution (1mL/L) as internal standard. A 4 points linear calibration curve was established before analysis of the real case samples. Solutions of 0 (blanc with internal standard), 1, 2.5 and 5mL/L of the solvent in 100mL H 2 O with 100µL of 1-butanol as internal standard were injected in split mode with a ratio of 40: 1. Samples with S/N >10 were dosed.
For pesticide determination, the EN 15662 method for pesticide analysis in foods of plant origin with QuEChERS extraction was adapted for analysis of the oils. The adaption consisted in taking 1g of sample instead of 10g. Cleanup was done using PSA -carbon black mix of 19:1 (w/w). The sample was mixed with 10mL acetonitrile, and shaken for 1minute. Then, a mixture of salts was added (4g MgSO 4 ; 1g NaCl; 1g sodium citrate and 0.5g disodium citrate sesquidydrate), and shaken for 1minute, centrifuged for 5minutes at 4000rpm. 1ml of the upper layer was mixed with the internal standard (triphenyl phosphate) and injected into the analytical systems. A total of 177 pesticides were scanned using LC/MS/MS or GC/MS/MS. LC-MS/MS analyses of pesticides were carried out on an Aquity ultra-performance liquid chromatography system (Waters, Zellik, Belgium). Chromatographic separation was achieved on an Aquity UPLC HSS T3 column (100mm x 2.1mm x 1.8µm). A linear multi-step solvent gradient was applied with the solvents A (water, 0.02% formic acid) and B (acetonitrile, 0.02% formic acid). The gradient consisted of 0-16min 100-0% (A); 16-18min, 100% (B); 18-20min, 0-100% (A). The flow was set to 0.4mL/min. The injection volume was 1µl. The temperature of the sample chamber was 15°C and the temperature of the column oven was set at 40°C. MS/MS measurements were carried out on a XevoTQS unit (Waters, Zellik, Belgium) in Multiple Reaction Monitoring (MRM) mode. MassLynx software (Waters) was used to detect pesticides in monitored data. Reported limit was at 0,1mg/ kg. Semi-quantitation of identified pesticides (propamocarb) was carried out in a second step with external calibration. CG/MS/MS analyses of pesticides were carried out on an Agilent 7000 Series triple Quad equipped with an HP-5MS (5% phenyl methyl siloxane) column (30m x 0.25mm x 0.25µm) in MRM mode. Screening of pesticides was done using Masshunter identification software. The reporting limit for all pesticides was 1mg/kg.
For metal determination the EN 13805:2014 (European Committee for Standardization, 2014) method was used. To 1g of oil in a PTFE vessel, 4mL of HNO 3 (65%), 1mL of H 2 O 2 (31%) and 1mL of H 2 O were added. The mixture was let to react for 5minutes at room temperature and then heated over a 30minutes' gradient to 180°C using a Mars 5X press microwave. This temperature was maintained over 30minutes. A volume of 1mL of the obtained solution was diluted with 1mL of internal standard (10ppb of Tb, Y and Sc in 5% HNO 3 ). Metal analyses (expect for mercury) were carried out in duplicate on an Agilent 7700x ICP-MS (Agilent Technologies, Waldbronn, Germany) in He collision cell modus and using external calibration (0.05-100µg/L). Mercury analyses were carried out on a Milestone DMA-80 (Milestone Srl, Sorisole, Italy) using 30mg of sample with following parameters: quadratic calibration at 254nm, temperature program: 200°C for 1min; elution: 650°C, 60s, amalgam heating: 650°C, 12s, measurement time 30s. LOD for all metals was 0.01µg/g. All vendors who informed about the batch number also informed about an expiration date, but only four vendors (16.7%) informed about all parameters listed in Table 1. No vendor discussed possible drug interactions. Drug-drug interactions or overdosage is generally not considered a serious health issue for CBD, but CBD is known to be an inhibitor of the cytochrome P450 enzyme family [15,16]. This may result in higher levels and even toxicity of other drugs if consumed together with CBD. CBD also reduces blood pressure [17] which may be a positive health effect in some cases but has to be considered when simultaneously taking anti-hypertensive drugs. In addition, CYP3A4 and CYP2C19 have been identified as major contributors to CBD metabolism. Inhibitors of these enzymes may increase CBD levels whereas inducers may reduce CBD blood levels. Consumers should be made attentive to these effects and be asked to discuss the CBD intake with their doctor.

Solvent content
Traditionally, extraction using ethanol with or without heating was the method used to prepare cannabis tinctures. This method has the advantage of being simple and cheap, but only limited control over extraction selectivity is possible. The process results in extracts with higher concentrations in side products (chlorophyll, pigments and tannins) and the need of extra work-up to eliminate the extraction solvent. Today, supercritical CO 2 extraction is the most popular preparation method. By controlling temperature and pressure, selective components of the plant material can be separated and collected from the bulk of the plant material. A total of 13 vendors gave information about the extraction method used. Out of them, five indicated cannabinoid extraction using a "raw" method, six indicated a CO 2 extraction method and one claimed the use of both "raw" method and CO 2 . Organic solvents were found in four samples: cannabis oil n°. 7 contained 2.70% of 2-propanol and oil n°. 15, 17 and 23 contained 1.05, 0.07 and 0.13% of ethanol, respectively. Probably, these four samples were prepared using traditional solvent extraction methods but none of the vendors informed about its use. Isopropanol is sometimes used to make tinctures for external use. Even if the concentrations found are low and do not pose a health risk, 2-propanol should not be used for the preparation of extracts intended for internal use as its major metabolite, acetone, is a central nervous system depressants [20].

Pesticide content
Pesticide regulation in commercial cannabis market is still a matter of debate [21]. Because cannabis remains an illegal plant in most countries, governmental regulations generally do not exist. In Europe the only notable exception is a Dutch regulation from 2002 containing policy guidelines for pesticides, herbicides and fertilizers stipulating that "All processes and procedures which may affect the quality of the product must be recorded in the documentation for each batch". While outdoor plantations generally do not need extensive pesticide or nutrient systems, indoor growers often use such products for pest control. Previous studies found fungicides, insecticides and acaricides in illegal cannabis plantations [22,23]. In our study, out of the 177 pesticides scanned, only propamocarb has been detected. It was semi-quantified in three samples: n°. 4 (1.5mg/kg), n°. 11 (2.0mg/kg) and n°. 22 (1.5mg/kg). Propamocarb is a fungicide of low toxicity, used among others against canker caused by Pleosporales fungi. The levels detected in the 3 samples do not pose any health risk.

Metal content
Metals detected in the CBD oil samples are summarized in Table  4. Cannabis is known for its easy uptake of metals from soil [24] which makes it a promising plant for bioremediation. In our study, all metal concentrations were below the limits fixed by European legislation (Commission regulation (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs): for cadmium (limit for vegetables: 0.05ppm), mercury (limit for fish as most contaminated food: 0.5ppm), tin (limit of inorganic tin in infant formula: 50ppm) or lead (limit in food complements: 3ppm). Only one sample (n°. 15) showed an abnormal high content of copper and zinc concentrations which may be explained by contamination during the extraction process using an organic solvent (ethanol) in copper containing vessles. However, these low toxicity elements are not posing serious health issue in this sample.

Conclusion
A total of 24 samples containing hemp extracts have been analyzed for cannabinoid, solvent, terpene, pesticide and metal contents. To the best of our knowledge, this is the first study to access user information and content control of commercial CBD oils. Most of the samples had substandard consumer information. Only four samples out of the 24 had overall information about expiration date, storage and dosage conditions, residual solvent content and possible side effects. All extracts contained CBD and/or CBDA but less than one third of the samples had total cannabidiol (CBD + CBDA) concentrations in the claimed concentration range. Pesticides were detected in 3 extracts and solvents in 4, all levels were in the non-toxic range. Also, all metal concentrations were below health concerns. There is no doubt that online commerce with cannabis-derived products (oil, powder, capsules, suppositories, chewing gum, soaps, skin lotions, cookies, ...) will become more and more popular as clinical evidence about its beneficial health effects for many different ailments keeps growing. Substandard products however, may, in the short term, be interesting for the vendors but, in the long term, may bring discredit to a highly beneficial product. However, not only producers and vendors are to blame as cannabis extracts are still in a legal grey zone. In most countries classification and consequently legalisation, are not clear and shifting somewhere between medication, herbal medicine, food supplement or novel food. Furthermore, some countries still register cannabis use offences, other have decriminalized its use, still others have increased penalties. Considering the impact, the products may have on consumer health, a harmonization of EU laws is highly desirable in order to clearly define its status and quality specifications, whether with or without psychotropic THC. Systematic testing of CBD extracts and all other cannabis-derived products should be mandatory for the benefice of the patients' health and honest CBD online retailers.